Dr. Chaim Wachtel

Introduction to PCR and qPCR Part II:

PCR!!

qPCR technical workflow

Sampling

DNA Extraction

RNA Extraction

DNase treatment

ReverseTranscription

qPCR DataAnalysis

Primer design

Primer design – key to successful PCR

• Good primer design saves time and money• Advanced applications require even more

stringent primer design– Multiplex– Low abundance

Good primer (pair) properties

Primers should have• 18-24 bases• 40-60% G/C • Balanced distribution of G/C and A/T bases• Tm that allows annealing at 55-65°C• No internal secondary structures (hair-pins)

Primer pairs should have• Similar melting temperatures, Tm , within 2-3 °C• No significant complementarity (> 2-3 bp)

– particularly not in the 3’-ends

´3´3

Cycling...

Sense

Antisense

Sense

Antisense

Sense

Sense

cAntisense

Antisense

The primer dimer (PD) problem

• Primers that interact are amplified by PCR.

• PD formation competes with the designed PCR and can compromise the reaction efficiency.

Solution to the PD problem

• Reduce the formation of PDs by– Good primer design (avoid 3’ complementarity)– Minimal annealing time– Good laboratory practice– HotStart– TouchDown

• Reduce the signal from PDs by

– Measuring fluorescence above the Tm of the PDs

– Use sequence-specific probe

Considerations

• Avoid targets with secondary structure• Avoid pseudogenes• Avoid genomic contamination by designing

primers to span intron-exon-junctions

exons

introns

PCR primers

Links for designing primers

• http://www.tataa.com/• http://www.ncbi.nlm.nih.gov/BLAST/• www.premierbiosoft.com/netprimer/netprl

aunch/netprlaunch.html• www.ensembl.org • http://www-genome.wi.mit.edu/cgi-bin/pri

mer/primer3_www.cgi• http://www.bioinfo.rpi.edu/applications/mf

old/dna/form1.cgi• Primer Design- Beacon Designer/AlelleID• Primer express 3 (AB)Primer express 3 (AB)

Primer Express

• Located on Software 1 • Easy to use• Not fool-proof, but none of them

are…..

Primer design-work flow

Find sequenceFind sequence Design PrimersDesign Primers

Primer3 or similarPrimer3 or similarsoftwaresoftware

Check PrimersCheck Primers

for desired for desired parametersparameters- Tm- Tm- amplicon size- amplicon size- secondary secondary structurestructure- complementarity- complementarity- specificity- specificity

Netprimer, BLAST Netprimer, BLAST and similar and similar softwaresoftware

Satis-Satis-factory?factory?

NoNo

Run PCRRun PCRYesYes

……and gel and gel electrophoresis to electrophoresis to check specificity check specificity and functionalityand functionality

NCBI or EnsemblNCBI or Ensembl

TaqMan Probe Design

• Amplicon size 70-150 bp• Tm of probe 68-70 °C• G/C content 30-70%• No G at the 5´end• Avoid runs of identical nucleotides• Avoid secondary structure• Avoid complementarity with primers• HPLC purification

Popular dyes and quenchers

• FAM• JOE• HEX• TET• VIC• ROX• Cy

• DABCYL• TAMRA• Black Hole Quenchers

RT-PCR

• Housekeeping genes– What are they– How do you choose

• Standard curve• Primer Dimer• Melt curve• Optimization• Test samples

Reference

Workflow – preliminary data analysis

Baseline settingsBaseline - is the initial cycles in PCR where there

is little change in fluorecence signal, usually cycle ~3-15

• Set the baseline• Fixed number of cycles • Adaptive baseline

• Control baselines in the linear scale (y-axis)

Raw data

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35

Cycles

cDNA 1:1cDNA 1:10cDNA 1:100

Baseline adjustment

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Cycles

cDNA 1:1cDNA 1:10cDNA 1:100

The different phasesExponential growth phase Plateau phase

Part of exponentialgrowth phase wheresignal > background(noise)

Samples must be compared in the exponential phase

0.01

0.1

1

10

100

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

cDNA 1:1

cDNA 1:10

cDNA 1:100

Threshold

Setting threshold• Purpose: Find a level of fluorescence where samples can be compared• The theoretical cycle where a sample intersect the threshold is called Ct

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1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

cDNA 1:1

cDNA 1:10

cDNA 1:100

Ct

Linear

scale

Logari

thm

ic s

cale

Threshold level

Ct valuesLogLog

Setting threshold

Ct (threshold cycle): Threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number

Setting threshold• Several methods available for threshold setting

– Standard deviation of the noisefor the first few cycles

– Second derivative maximum (SDM)– Best fit of standard curve (highest r2)– Manual setting

A two-fold difference in copy number should have one Ct difference no matter where the threshold is set within the exponential phase

Dilution series and standard curves

• Used to control the quality of your assays• Absolute quantification

– Standards = Diluted templates of known concentration

– Standard curve = Ct of each standard sample is plotted against the known concentration

– Used to determine concentrations of unknown samples

– Absolute quantification is dependent on the quality of the standard curve

Standard curve

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100

2.00E+092.00E+082.00E+072.00E+062.00E+052.00E+042.00E+03

Comment: Always cover the whole range of sample concentrations.

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1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09

Concentration (log scale)

Interpretation of the standard curve

• Linear regressionY = ax + b

a = slope that givesefficiency of PCRfrom 10–1/a = 1 + efficiency

b = # of cycles for detecting one molecule

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1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09

Concentration (log scale)

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1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09

Concentration (log scale)

Relative quantification• Often there is no good standard available• Compare amount with reference

• Reference genes• Genomic DNA• Spike• Ribosomal RNA

• Example– Expression of target gene is 10% of the expr. of

housekeeping gene.– Same gene in other tissue, expression is 100%.

Comparing treatments

MIQUE Nomenclature

• MIQUE - Minimum Information for Publication of Quantitative Real-Time PCR experiments

Suggested nomenclature

• Reference genes not housekeeping genes• Quantification not quantitation• Hydrolysis probes not TaqMan probes• Quantification cycle Cq replaces Ct, Cp, TOP

Melting curve analysis• Melting curves are obtained by measuring

the fluorescence while increasing temperature

• Use a dye binding to double stranded DNA

70 95 Temp80 90

Melting curve analysis

• Confirms formation of the expected product (each dsDNA has its characteristic melting temp Tm)

• Distinguishes between specific PCR products and non-specific products (e.g. primer-dimers)

• High resolution melt – mutation and methylation analysis

Melting curve

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65 70 75 80 85 90 95Temperature

cyp3a high 1:1000cyp3a NTC

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65 70 75 80 85 90 95Temperature

cyp3a high 1:1000cyp3a NTC

Melting temperature Tm is characteristic of the %GC, length and sequence. The product can be identified from the Tm.

Tm = 90 °C

Tm = 81.5 °C

derivat

ive1st

4-steps PCR 4-steps PCR can be used to eliminate primer-dimer

signals

40 cycles

Example – 4 steps PCR

100% efficiency 75% efficiency

100% efficiency 90% efficiency

80% efficiency 50% efficiency

100%

90% 80% 75% 50%

GMNv1

3.305

3.823

4.456

4.826 7.47

VN1 3.186

3.451

3.775

3.924 4.991

Ratio 1.04 1.11 1.18 1.23 1.5

Reference primer efficiency

RT-PCR –testing samples

• ALWAYS perform melt curve• ALWAYS run negative controls

– No RT– No template

• Always Always Always run standard curve

• Triplicate of each sample!!

Requirements for RT-PCR Experiment

• Always perform standard curve• All samples in triplicate• NTC control• No RT control• Prepare mix without cDNA; add this to each tube separately• Divide plate by gene and not sample• Do not need reference gene on every plate• Melt Curve• Check RNA- otherwise don’t bother with experiment• Do not rely on only 1 reference gene- check more than one

per project• Every project is different!• Don’t be afraid to ask me questions, especially BEFORE

starting the project.

Digital PCR

From Relative quantity to absolute quantity

Commercially available machines

Fluidigm

QuantaSoft (Life Technologies)

Rain Dance

Bio Rad QX100